Growth factors are important for normal developmental processes, as well as for healing of wounds. Their abnormal expression has been implicated in neoplasia and other proliferative disorders. The kringle-containing protein hepatocyte growth factor (HGF) was originally identified as a potent growth factor involved in liver regeneration after liver injury or partial hepatectomy. It is now known that HGF functions as a growth factor for a broad spectrum of tissues and cell types. In addition, it has been recently discovered that HGF is identical to scatter factor (SF) a cytokine secreted from certain fibroblasts that enhances movement and causes the dissociation and scattering of epithelial cells (Gheradi & Stoker, 1990). The proto-oncogene c-met, a tyrosine kinase, has been found to be the cell surface receptor for HGF (Rubin et al., 1991; Bottaro et al., 1991). These properties may be important for metastasis of tumor cells.
In 1973 it was recognized that serum from partially hepatectomized rats stimulated hepatocyte proliferation in vitro (Morley et al., 1973). One of the agents responsible for this phenomenon was identified and isolated from such serum and from serum of patients with fulminant liver failure (Morley et al., 1973; Michalopoulous et al., 1984; Nakamura et al., 1984; Gohda et al., 1988). This agent was named hepatopoietin A or hepatocyte growth factor (HGF). HGF stimulates hepatocyte DNA synthesis and proliferation. Its serum concentration increases dramatically after rats undergo partial hepatectomy and decreases when the liver regenerates. HGF is produced by non-parenchymal liver cells (Schirmacher et al., 1992) and acts directly on hepatocytes in a paracrine fashion to stimulate cell multiplication. Although HGF stimulates growth of normal hepatocytes, it also has antiproliferative effects on hepatocarcinoma cells in culture (Tajima et al., 1991; Shiota et al., 1992).
HGF is a heterodimer of 82 kD composed of a .alpha.- and .beta.-subunit with 51 kD and 26 kD molecular weight, respectively. The cDNAs for human and rat HGF have been cloned and characterized by several groups (Miyazawa et al., 1989; Nakamura et al., 1989; Okajima et al., 1990; Seki et al., 1990; Tashiro et al., 1990; Rubin et al., 1991).
HGF has no obvious homology with other known growth factors but is 38% homologous to plasminogen. It contains four kringle domains followed by a serine protease-like domain where the active site His and Ser have been changed to Gln and Tyr, respectively. HGF has no detectable protease activity. At present the function of the kringle domains in HGF is unknown.
Kringle domains were first identified in bovine prothrombin as an internal duplication of a triple-disulfide-bonded structure containing approximately 80 amino acids (Magnusson et al., 1975). Kringle domains were until recently only characterized in plasma proteins that functioned in blood coagulation or fibrinolysis (Davie et al., 1986) which includes prothrombin, Factor XII, urokinase-type plasminogen activator, tissue-type plasminogen activator and plasminogen. Recently, apolipoprotein(a) and HGF have also been shown to contain kringle domains. Apolipoprotein(a) is thought to be involved in atherosclerosis (McLean et al., 1987). Kringle structures are thought to function autonomously (Trexler & Patthy, 1983; van Zonneveld et al., 1986) and fold independently (Tulinsky et al., 1988).
Kringles appear to be protein-binding domains and have been shown to be essential for the function of prothrombin, plasminogen and tissue plasminogen activator. The functions of all other kringle structures has not been determined, but since these structures are over 50% identical with each other, it is reasonable to assume that they are involved in binding interactions with other proteins essential for their regulation.
Two functional variants of HGF have been identified and have been found to be expressed at variable levels depending on the cell line or tissue being analyzed. A form of HGF containing the amino-terminal end of the protein including the first two kringle domains appears to result from alternative processing of the gene coding for HGF (Chan et al., 1991; Miyazawa et al., 1991). This variant binds to the c-met receptor although not as effectively as the full-length protein. Another variant has a five amino acid deletion in the first kringle domain that appears to have no effect on its activity (Seki et al., 1990; Rubin et al., 1991). Specific domains in HGF have been deleted by using techniques in molecular biology and the resultant proteins have been studied in various assays where native HGF can be measured. Matsumoto et al. (1991) concluded that the amino-terminal portion of the protein including the first and second kringle domains are essential for biological activity of HGF and possibly binding to the receptor.
Chromosomal abnormalities in a number of neoplastic diseases are sometimes associated with the activation of a proto-oncogene or the loss of a gene that suppresses tumor growth. Growth factors are important for normal developmental processes, as well as healing of wounds. Their abnormal expression has been implicated in neoplasia and other proliferative disorders (Aaronson, 1991). Growth factors are involved in signaling pathways that influence normal cellular differentiation. These proteins cause cells in the resting phase (Go) to enter and progress through the cell cycle. oncogenic mutations in several growth factors result in unregulated cell growth. Tumor suppressor genes are genes expressed in normal cells that play regulatory roles in cell proliferation, differentiation and other cellular events. Loss or inactivation of these genes is oncogenic. Tumor suppressor genes that have been extensively characterized include the genes for colon carcinoma, retinoblastoma, type 2 neurofibromatosis, the genes involved in Wilms tumor and the p53 gene (reviewed in Weinberg, 1991). Tumor suppressor genes are involved in cell cycle control, signal transduction, angiogenesis, and development (Sager, 1989; Weinberg, 1991).
The concept that the loss of genetic material or the inactivation of a gene plays an important role in human cancer is based on the original observation that somatic cell hybrids between tumor cells and normal cells were no longer tumorigenic. This indicated that normal cells contain genes coding for tumor suppressors whose function was absent in cancer cells. In addition, cytogenic and restriction fragment length polymorphism (RFLP) analyses have established an association between the loss of genetic material on specific chromosomes and the development of various human malignancies.
Deletion of the short arm of human chromosome 3 has been implicated in small cell lung carcinoma (SCLC; Whang-Peng et al., 1982; Naylor et al., 1987), other lung cancers (Kok et al., 1987; Brauch et al., 1987), renal cell carcinoma (Zbar et al., 1987; Kovacs et al., 1988) and yon Hippel-Lindau syndrome (Seizinger et al., 1988) which suggests that one or more tumor suppressor genes reside on chromosome 3p which manifest their transformed phenotype upon their inactivation. The chromosomal locus DNF15S2 (also called D3F15S2) is a RFLP probe that most consistently is associated with loss of heterozygosity in SCLC, being detected in virtually 100% of SCLC.
Lung cancer is a common human malignancy with 150,000 new cases reported each year in the United States. Unfortunately, 90% of affected persons will die within 5 years of diagnosis. Mortality due to lung cancer has increased more than 15% since 1973. Increases in cigarette smoking from 1900 until the early 1960s has transformed lung cancer from a rare disease at the turn of the century to the current leading cause of cancer death. In women, lung cancer surpassed breast cancer as the leading cause of cancer death in 1986 with rates expected to continue to increase for at least another ten years (Henderson et al., 1991).
Lung cancer is divided into small cell and non-small cell varieties. The non-small cell lung cancers include adenocarcinoma, squamous and epidermoid lung cancer and large-cell lung cancer. Chromosome 3p(14-23) changes have been found in nearly all small cell lung cancers and in a large fraction of non-small cell lung cancers.
Cancer of the kidney accounts for 1-2% of all malignancies (excluding skin cancer) with renal cell carcinoma comprising 85% of these. Renal cell carcinoma (RCC) occurs in sporadic and familial forms and are commonly seen in the age group between 50 to 70 years. Cigarette smoking is a known risk factor for this form of cancer (Walter et al., 1989). Deletion of the short arm of chromosome 3 is the most commonly involved region of the genome in RCC and therefore appears to play a role in the development and/or progression of this form of cancer.
Several genes have been localized near or at the D3F15S2 locus. The ER.beta.EAB locus coding for a DNA-binding thyroid hormone receptor is localized to human chromosome 3p21-25, and overlaps deletions found in SCLC. Leduc et al. (1989) determined that many non-SCLC tumors retained both ERBAB alleles while the D3F15S2 locus was reduced to homozygosity, ruling out a role for the thyroid hormone receptor in this form of cancer. The gene encoding aminoacylase-1 at 3p21 is inactivated in a large fraction of SCLC (Naylor et al., 1982, 1989). A similar allelic loss is observed in sporadic renal cancers and there are cytogenetic abnormalities of this region in familial renal cell cancer. The gene coding for protein-tyrosine phosphatase.gamma. (PTP.gamma.) maps to 3p21 (LaForgia et al., 1991). This protein and homologous family members reverse the effect of protein tyrosine kinases, of which, some have been identified as oncogenes (ie., met, fms, kit, ERBB). In one study, one PTP.gamma. allele was deleted in 3 of 5 renal carcinoma cell lines and in 5 of 10 lung carcinoma samples tested (LaForgia et al., 1991). In summary, the key gene(s) responsible for tumor suppressor activity at this locus is unknown, although there are some candidate genes.